In rat brain, high-conductance Ca²⁺-activated K⁺ (BK) channels are targeted to axons and nerve terminals [Knaus, H. G., et al. (1996) J. Neurosci. 16, 955−963], but absolute levels of their regional expression and subunit composition have not yet been fully established. To investigate these issues, an IbTX analogue ([¹²⁵I]IbTX-D19Y/Y36F) was employed that selectively binds to neuronal BK channels with high affinity (K_d = 21 pM). Cross-linking experiments with [¹²⁵I]IbTX-D19Y/Y36F in the presence of a bifunctional reagent led to covalent incorporation of radioactivity into a protein with an apparent molecular mass of 25 kDa. Deglycosylation and immunoprecipitation studies with antibodies raised against α- and smooth muscle β-subunits of the BK channel suggest that the β-subunit that is associated with the neuronal BK channel is a novel protein. Quantitative receptor autoradiography reveals the highest levels of BK channel expression in the outer layers of the neocortex, hippocampal perforant path projections, and the interpeduncular nucleus. This distribution pattern has also been confirmed in immunocytochemical experiments with a BK channel-selective antibody. Taken together, these findings imply that neuronal BK channels exhibit a restricted distribution in brain and have a subunit composition different from those of their smooth muscle congeners.